The invention relates to a method for the production of natural L-cysteine by fermentation.
The amino acid L-cysteine is used for example as a food additive (particularly in the baking industry), as a feed stock in cosmetics, and also as a starting material for producing pharmaceutical active ingredients (in particular N-acetylcysteine and S-carboxymethylcysteine) and is therefore of economic importance.
L-Cysteine plays a key role in sulfur metabolism in all organisms and is used in the synthesis of proteins, glutathione, biotin, lipoic acid, methionine and other sulfur-containing metabolites. Moreover, L-cysteine serves as a precursor for the biosynthesis of coenzyme A. The biosynthesis of L-cysteine has been investigated in depth in bacteria, particularly in Enterobacteria, and is described in detail in Kredich (1996, Biosynthesis of cysteine, p. 514-527. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.).
Besides the classical production of L-cysteine by means of extraction from keratin-containing material such as hair, bristles, horns, hooves and feathers or by means of biotransformation by enzymatic conversion of precursors, a method for producing L-cysteine by fermentation was also developed some years ago. The prior art with respect to the production of L-cysteine by fermentation using microorganisms is described in detail e.g. in U.S. Pat. No. 6,218,168B1, U.S. Pat. No. 5,972,663A, US2004038352A2, CA2386539A2, EP1769080 and EP2138585. The bacterial host organisms used here are inter alia strains of the genus Corynebacterium and representatives from the family of the Enterobacteriaceae, such as e.g. Escherichia coli or Pantoea ananatis. 
Besides the classic procedure for arriving at improved L-cysteine producers by mutation and selection, targeted genetic modifications to the strains have also been performed in order to achieve an effective L-cysteine overproduction.
For example, the insertion of a cysE allele coding for a serine O-acetyl transferase with a reduced feedback inhibition by L-cysteine led to an increase in cysteine production (U.S. Pat. No. 6,218,168B1). As a result of a feedback-resistant CysE enzyme, the formation of O-acetyl-L-serine, the direct precursor of L-cysteine, is largely decoupled from the L-cysteine level in the cell.
O-Acetyl-L-serine is formed from L-serine and acetyl-CoA. Consequently, the provision of L-serine in a sufficient amount for L-cysteine production is of great importance. This can be achieved by introducing a serA allele coding for a 3-phosphoglycerate dehydrogenase with a reduced feedback inhibition by L-serine. As a result, the formation of 3-hydroxypyruvate, a precursor of L-serine, is largely decoupled from the L-serine level in the cell. Examples of such SerA enzymes are described in EP0620853, U.S. Pat. No. 7,582,460B2 and EP0931833.
Moreover, it is known that the L-cysteine yield in the fermentation can be increased by weakening or destroying genes coding for L-cysteine-degrading enzymes, such as e.g. the tryptophanase TnaA or the cystathionine-β-lyases MalY or MetC (EP1571223).
The increase in the transport of L-cysteine from the cell is a further way of increasing the product yield in the medium. This can be achieved by overexpression of so-called efflux genes. These genes code for membrane-bound proteins which mediate the export of L-cysteine from the cell. Various efflux genes for the L-cysteine export have been described (U.S. Pat. No. 5,972,663A, US2004038352A2, US2005221453, WO2004113373).
The export of L-cysteine from the cell into the culture medium has the following advantages:    1) L-Cysteine is continuously removed from the intracellular reaction equilibrium, with the result that the level of this amino acid in the cell is kept low and consequently the feedback inhibition of sensitive enzymes by L-cysteine does not occur:L-cysteine (intracellular)⇄L-cysteine (medium)  (1)    2) The L-cysteine released into the medium is oxidized to the disulfide L-cystine in the presence of oxygen, which is introduced into the medium during the culturing (U.S. Pat. No. 5,972,663A):2 L-cysteine+½O2⇄L-cystine+H2O  (2)            Since the solubility of L-cystine in aqueous solution at a neutral pH is only very low, especially compared to L-cysteine, the disulfide precipitates out even at a low concentration and forms a white precipitate:L-cystine (dissolved)⇄L-cystine (precipitate)  (3)        By virtue of the precipitation of L-cystine, the level of the product dissolved in the medium is reduced, as a result of which the reaction equilibrium of (1) and (2) in each case is also drawn to the product side.            3) The technical complexity for purifying the product is considerably lower if the amino acid can be obtained directly from the culture medium than if the product accumulates intracellularly and a cell disruption has to be performed first.
During the oxidation of two molecules of L-cysteine, the disulfide L-cystine is formed. This reaction is reversible, which means that L-cystine can be converted back to L-cysteine by reduction. If, after being separated off from the cells (e.g. using a decanter), L-cystine is reduced to L-cysteine again by means of electrolysis, then this chemical conversion means that such L-cysteine cannot be declared as natural according to the flavorings regulation. According to the EU flavorings regulation (1334/2008 Article 22 of the regulation for reforming the labeling regulations under the Foodstuff Law), natural flavorings are defined as follows: “natural” flavorings are chemically defined substances with flavoring properties which occur naturally and have been found in nature. They are obtained by suitable physical, enzymatic or microbiological processes from plant, animal or microbiological starting materials, which are used as such or processed for human consumption by means of one or more conventional food preparation processes (including microbiological processes such as fermentation, see Annex II).
The term “natural” is also used in this sense in the present application. There is great interest in the use of natural raw materials in the manufacture of flavorings. However, there has hitherto been no process for producing natural cysteine by fermentation.
There is a series of compounds which are able to catalyze the oxidation of SH groups; for example, it is known that heavy metal salts such as iron salts or zinc salts are essential additives in fermentations and that these components are able to effectively catalyze the oxidation of cysteine to cystine (US2008190854A2).
U.S. Pat. No. 6,218,168B1 describes that the fermentation medium for the production of L-cysteine and its derivatives comprises an iron concentration of 15 mg/l (75 mg/l iron sulfate heptahydrate). EP1389427A1 discloses a medium for the production by fermentation of L-cysteine, L-cystine and thiazolidine, in which the iron concentration is given as 14.9 mg/l (74 mg/l iron sulfate heptahydrate).
US2010/0093045A1 describes a medium for the production of L-cysteine which comprises only 2 mg/l iron (10 mg/l iron sulfate heptahydrate). However, this medium is used only for use in shake flasks on a laboratory scale (media volume 20 ml) and not for fermentation in a production fermenter. EP2133429A1 mentions a culture medium for the production of L-cysteine, L-cystine, its derivatives or a mixture thereof which comprises only 0.34 mg/l iron (1.7 mg/l iron sulfate heptahydrate). This medium is also not described for a use in fermentation, but for culturing in small tubes (media volume 2 ml). Usually, under these culturing conditions (batch culture, poor oxygen supply, no pH regulation) only very low cell densities (ca. 0.5 to 2 g/l dry biomass) and small product yields are achieved. This is evident by reference to the L-cysteine yields achieved in shake flasks or small tubes of 0.25 g/l (US2003/0077766A1), 0.3 g/l (EP2133429A1) and 1.2 g/l (US2010/0093045A1).
In production fermenters, however, high cell densities are desired in order, as a consequence of the biomass-specific product formation rates, to achieve correspondingly high volumetric yields which only permit an economical process on an industrial scale. The quality of an industrial process for microbiological material production is generally assessed by reference to the productivity. This parameter describes the total amount of product formed per liter of medium per fermentation time.
One disadvantage of the described methods for the production of L-cysteine by fermentation is that the amino acid is present in the culture broth in various forms. In addition to the precipitated L-cystine in the precipitate, soluble L-cysteine, but also L-cystine in dissolved form and thiazolidine are found in the culture supernatant (U.S. Pat. No. 6,218,168B1, U.S. Pat. No. 5,972,663A, CA2386539A2). This thiazolidine (2-methylthiazolidine-2,4-dicarboxylic acid) is the condensation product of L-cysteine and pyruvate, which is formed in a purely chemical reaction.
Within the context of this invention, the term “total cysteine” includes L-cysteine and the L-cystine and thiazolidine compounds derived therefrom and which are formed during the fermentation and accumulate in the culture supernatant and in the precipitate.
In the known methods, the composition of the total cysteine varies at the end of the fermentation: the fraction of precipitated L-cystine is between 40-66% (U.S. Pat. No. 5,972,663A, CA2386539A2), the remaining 34-60% are present in the culture supernatant in the form of soluble products predominantly as L-cysteine and thiazolidine. This product heterogeneity hinders the recovery and purification of the target product natural L-cysteine from the culture broth.
A method would therefore be desirable in which the end product produced is predominantly soluble L-cysteine. Moreover, as far as possible no thiazolidine should be formed. The purification of the target product L-cysteine from the culture supernatant would be considerably easier with such a process since the L-cystine present as precipitate can be separated off together with the cells by a simple separator step, and the soluble L-cysteine can be isolated by ion exchange adsorption.